Immune Checkpoint Molecules B7-H6 and PD-L1 Co-Pattern The Tumor Inammatory Microenvironment: Associations and Clinical Signicance in Human Breast Cancer

B7-H6 and PD-L1 belong to the B7 family co-stimulatory molecules ne-tuning the immune response. This report exposes for the rst time the clinical implication of B7-H6 protein expression in relation with PD-L1 status and Natural Killer Cells inltration as potential biomarkers in tumor inammatory microenvironment. Herein, we explore the expression levels of B7-H6 protein by cancer cells and immune inltrating cells in human breast cancer tissues and evaluate their associations with PD-L1 expression, NK cell status and clinical pathological features as well as the prognosis. Immunohistochemistry labeling method was used to assess B7-H6 and PD-L1 proteins expression by cancer and immune cells. Associations between immune checkpoint, major clinicopathological variables and survival rates were analyzed. B7-H6 protein was revealed in both breast and immune cells. Tumor B7-H6 expression is highly associated with Her2 over expression. B7-H6 + immune cells are highly related to SBR grade and associated with PD-L1 and NK cells status. Survival analysis showed that patients with low expression of B7-H6 by cancer cells had better prognosis. Conversely, B7-H6 + immune cells were signicantly associated with longer survival. Our result strongly suggests an interaction between B7 molecules that contributes to a particular design of the inammatory microenvironment. This may inuence the eciency of therapies based on antibodies blocking the PD-L1/PD1 pathway and can explain why the clinical benets are seen only in a fraction of patients treated with immune checkpoint inhibitors. immunostaining in BCC, H-scores were generated as described in methods. In order to investigate the correlation between clinical parameters and the B7-H6 and PD-L1 protein expression levels in BCC, we categorized the 156 patients into two major subgroups according to the intensity of B7-H6 and PD-L1 immunohistochemical staining. Our data show that B7-H6 BCC expression levels were low in 61% (0 ≤ H-score < 100) and high in 39% of cases (H-score ≥ 100). For PD-L1 BCC distribution in our cohort, our results show that PD-L1 BCC expression levels were low in 80% (0 ≤ H-score < 100) and high in 20% of cases (H-score ≥ 100). Regarding TILs densities distributions, TILs-B7-H6 + were predominantly localized in the stroma surrounding BCC clusters and abundant in 14% of cases, conversely, TILs-PD-L1 + were abundant in 60% of cases. the cellular membrane or sequestered in the cytoplasm or even secreted. These concepts justify the complexity in understanding the mechanisms controlling the tumor inammatory microenvironment through B7 molecules. Taking into account our nding from the present study, we can suspect a protective role of B7-H6 and PD-L1 molecules when co-expressions are manifested on lymphocytes inltrating tumors, probably these cells produce interleukins such INF-γ which modulate NK cells function. To better elucidate the immunomodulatory mechanism of B7 molecules, it is important to combine more precise analyzes of their location and to conduct analyzes allowing biomarkers co-revelation. Therefore, immunotherapy targeting checkpoint molecules must be revised. The use of combined immune checkpoint inhibitors may result in reduction of tumor burden and patient benet in some cases. In others situations probably it cannot be effective because of the double role of these two molecules, targeting other molecules from the B7 family can be an important axis which deserves further investigations to decipher their contribution in innate immunity monitoring, probably, we will need to target several molecules at the same time and use targeted therapy according to the patient characteristics. performed to assess the using Chi-square tests for correlation for quantitative variables and ANOVA test for quantitative variation among classes of qualitative variables. The cumulative survival (overall survival, OS; recurrence-free survival, RFS) times were calculated using the Kaplan–Meier method and compared with the log-rank test. A cox-regression was also performed in order to evaluate the signicance of prognostic factors on survival in a multivariate context (adjusting for confounding variables). All p-values less than 0.05 were considered signicant.


Introduction
Breast cancer is the most diagnosed malignancy in women worldwide and is a highly heterogeneous disease presenting a broad range of molecular and clinical characteristics [1]. Towards this heterogeneity, the establishment of an effective immune response requires the participation of several actors taking into account this variability. Both adaptive and innate immunity contribute to the immune editing of malignancy features [2,3]. But more attention is devoted to natural immunity particularly to Natural killer (NK) cells which are important components of the innate immune system and play a central role in tumor microenvironment (TME) designing [4] since they participate in adaptive response through their cytokine secretion polarizing T-cell activation [5][6][7].
Moreover, the modulation of NK cells response can be controlled by the immune checkpoints molecules from the B7 family [8]. The most known immune checkpoint regulators are programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) and CTL antigen 4 (CTLA-4), which are highlighted in a variety of cancers and their therapeutic advances have encouraged researchers to investigate other targets from the B7/CD28 family. Recently, ve new B7 family ligands, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7, were identi ed. These later are increasingly investigated in various solid cancers looking for signi cant association with cancer progression and patient's prognosis in different clinical retrospective studies [9][10][11][12].
Among this group, B7-H6 seems to be a potential target for new immunotherapy strategy. B7-H6 (also known as NCR3LG1) is a ligand for NK-cell-activating receptor NKp30 [13]. Human B7-H6 protein is rarely expressed in normal tissues, but in contrast, is overexpressed in various primary human tumors, including leukemia, lymphoma, and gastrointestinal stromal tumors [14]. However, B7-H6 can be induced at the surface of CD14(+)/CD16(+) proin ammatory monocytes and neutrophils upon stimulation by ligands of Toll-like receptors or proin ammatory cytokines such as IL-1β and TNF-α [15]. B7-H6 binds to NKp30 through the complementarity-determining region (CDR)-like loops of its V-like domain in an antibody-like interaction [16]. NK cells eliminate tumor cells expressing B7-H6 directly by cytotoxicity through co-signals balancing between activators and inhibitors ones, or indirectly by cytokines release. Hence the expression of B7-H6 by tumor cells is an important mechanism involved in activation of innate immunity mediated by NK cells. But to divert this visibility to the immune system, the malignant cells release or hide the B7-H6 molecule to prevent NK-mediated recognition [17].
Regarding the clinical signi cance of B7-H6 expression in human cancers, it has been provided in few studies. In ovarian cancer, positive B7-H6 staining was predominantly observed on the membrane and in the cytoplasm of the ovarian cancer cells. The overall survival rate of the subgroup with lower B7-H6 expression was signi cantly better compared to those with higher B7-H6 expression [18]. In astrocytoma, B7-H6 positive expression was signi cantly associated with World Health Organization Grade [19]. Recently, high expression of B7-H6 has been proven to be a predictor of poor prognosis in esophageal squamous cell [20]. In the case of breast cancer disease, B7-H6 expression was revealed to be an unfavorable prognosis biomarker [21].
Despite these established expression in cases of human solid cancers, the association between B7-H6 and PD-L1 expression in human cancer remains unknown. In the current study, we focused on determining the co-expression pro les and clinical signi cance of B7-H6 and PD-L1 in women breast cancer, we assessed especially the contribution of B7-H6 and PD-L1 expression by tumor cells and immune cells colonizing tumor microenvironment. We also examined and compared their relation with NK cells status in controlling patient's survival.

B7-H6 and PD-L1 immunodetection in breast cancer
The immunohistochemistry analysis showed positive staining for B7-H6 (Fig. 1a) and PD-L1 (Fig. 1b) both on the membrane and in the cytoplasm of cancer cells, but also in TILs ( Fig. 1a and 1b). Representative examples of B7-H6 and PD-L1 immunostaining in breast cancer cells (BCC) and TILs are shown in Fig. 1C and 1D respectively. B7-H6 and PD-L1 expression were evaluated successfully by IHC in 156 BC tissues and positive controls at differential extent and intensity (Fig. 1c). Based on the extent and intensity of cyto-membranous immunostaining in BCC, H-scores were generated as described in methods. In order to investigate the correlation between clinical parameters and the B7-H6 and PD-L1 protein expression levels in BCC, we categorized the 156 patients into two major subgroups according to the intensity of B7-H6 and PD-L1 immunohistochemical staining. Our data show that B7-H6 BCC expression levels were low in 61% (0 ≤ H-score < 100) and high in 39% of cases (H-score ≥ 100). For PD-L1 BCC distribution in our cohort, our results show that PD-L1 BCC expression levels were low in 80% (0 ≤ H-score < 100) and high in 20% of cases (H-score ≥ 100). Regarding TILs densities distributions, TILs-B7-H6 + were predominantly localized in the stroma surrounding BCC clusters and abundant in 14% of cases, conversely, TILs-PD-L1 + were abundant in 60% of cases.
The correlation between patient's clinical parameters and B7-H6/PD-L1 expression has been shown in Table 1. Statistical analyses show a signi cant correlation of B7-H6 BCC expression with only Her-2 expression (p < 0.01) and molecular subtypes (p < 0.01). However, we did not nd any correlation with SBR grade, tumor size, lymph node invasion or metastasis. For correlation of TILs-B7-H6 status with clinicopathological parameters, we found signi cant relation with lymph node status (p < 0.05), ER status (p < 0.01) and SBR grading (p < 0.001). Regarding PD-L1 expression, our results display a signi cant correlation of PD-L1 BCC expression with histological type (p < 0.05) and SBR grade (p < 0.001), but no correlation between patient's clinical parameters and TILs-PD-L1 status was found except of a limited signi cant correlation with SBR grade (p = 0.049).

Correlations of B7-H6 expressions with PD-L1 and NK-TILs status
To look for eventual role of B7-H6 in in ammatory tumor microenvironment designing, we investigated the relationship of B7-H6 expression with PD-L1 and NK-TILs status. For more ascertainment, we conducted two types of analyzes. The rst way evaluates the relations on the raw results either in H-scores for the BCC expression or in TILs scores shared in 3 classes, as it was described in material and method. Data of these analyzes are shown in Fig. 2. The second way evaluates the relationships between the different parameters after the classi cation into two classes according to the cut-off mentioned in material and method and based on other previous works [18], [22]. Analyses based on raw data show signi cant correlations of PD-L1 BCC expression with the status of NK-TILs (p < 0.001), TILs-B7-H6 (p < 0.001), and TILs-PD-L1 (p < 0.001) (Fig. 2a-c). In contrast, a poor signi cant correlation of B7-H6 BCC expression with the status of TILs-PD-L1 (Fig. 2d, p = 0.0318) and NK-TILs (Fig. 2f, p = 0.0289) and insigni cant correlation with TILs-B7-H6 (Fig. 2e) Table).

Prognostic signi cance of B7-H6 expression
In order to look for the prognostic value of B7-H6 expression in women breast cancer, the log-rank survival analyses were performed according to B7-H6 expression levels in BCC or TILs scores after classi cation and collection of survival data. As shown in (Fig. 3a), patients with low B7-H6 BCC expression had a markedly longer OS compared to patients with high B7-H6 expression, however DFS curves are inverted but almost confused (Fig. 3b). In regards to TILs-B7-H6 status in survival analysis ( Fig. 3c and d), both OS and DFS rates of the subgroup with higher TILs-B7-H6 + are longer than those of the subgroup with lower TILs-B7-H6+, Kaplan-Meier survival curves for OS show signi cant differences between the two groups (p = 0.017) but no signi cance with DFS curves (p = 0.128). We then addressed the prognostic relevance of B7-H6 expression by tumor cells or TILs, we investigated survival data among combined groups; group 1 = TILs-B7-H6 Low /B7-H6 BCC Low , group 2 = TILs-B7-H6 High /B7-H6 BCC Low/High and group 3 = TILs-B7-H6 Low /B7-H6 BCC High . As shown in Fig Prognostic signi cance of B7-H6 besides others immune in ammatory biomarkers in Her2 positive cancer samples In order to better understand the impact of B7-H6 and others immune chokepoint molecules within Her2 positive cancer subtype, we sought correlations between clinical parameters and B7-H6 or PD-L1 expressions by tumor cells and TILs (Table 2), statistical analysis show only signi cant associations of B7-H6 or PD-L1 BCC expression with SBR grade (p = 0.025 and p = 0.035 respectively) but a limited signi cant association between TILs-B7-H6 status and lymph node invasion (p = 0.055). Furthermore associations between immunological parameters were performed ( Supplementary Fig. 1 Fig. 2a-c). In contrast, no difference in OS curves was observed in high or low TILs-PD-L1 presence but patients with high PD-L1 BCC expression tended to have a better survival (supplementary Fig. 2d-f).

Discussion
It remains challenging to build knowledge about the dynamism of the in ammatory tumor microenvironment and to understand the behavior of the main intersecting mediators. A growing number of studies have shown that in ammation plays a critical role in tumorigenesis [23]. The in ammatory tumors microenvironment is characterized by the presence of host leukocytes both in the supporting stroma and in tumor areas [24]. These leukocytes are commonly called Tumor-in ltrating lymphocytes (TILs) which are essential for establishing an immune antitumor response but they may contribute to cancer growth and immunosuppression associated with malignancy [34,35]. TILs can be adaptive immune cells or innate immune cells such as cytotoxic cells particularly NK cells which we are interested in studying in our cancer research elds. Moreover, it is clear that in ammatory status involves several molecules such as immune checkpoints. These molecules participate mutually but differently in cellular response mediated by NK cells, but the pro le of these molecules is not well understood. Members of the B7 family have been shown to be important participants in the TME designing notably B7-H1 (or PD-L1) molecule was widely studied in breast cancer and others solid tumors [27]. Others are less studied such as B7-H6 molecule which appears to be of importance. In this study, we have described the expression of B7-H6 and PD-L1 both on cancer cell and TILs in women breast carcinoma. Our results show for the rst time the expression of B7-H6 by TILs suitably within solid tumors. We have analyzed the widest part of the biopsy because we are convinced that TMAs tissue formats might not provide su cient representation of the TME notably, immune in ltrating cells. Previous studies have shown only the expression of B7-H6 by cancer cells but no study have reported their expression by immune cell when analyzes are performed on TMA tissues [18,21], [37]. So, one must be vigilant in certain technical aspects which can limit or even distort results, this point was raised previously by Sobral-Leite and colleagues [29].
Our study is the rst to investigate the concomitant pro le of B7-H6 and PD-L1 expression in breast carcinoma tissues by exploring their relationship rst, and then their implication in innate immunity through their associations with NK cells status, and nally by looking for their combined prognostic value. Our results have shown that the two immune checkpoint molecules of interest participate differently in breast carcinoma physiopathology despite the strong association between them. Originally, our data have shown that the biomarker B7-H6 has a different clinical signi cance depending on its expression either by tumor cells or by in ltrating immune cells. In fact, statistical analysis demonstrated that high levels of B7-H6 in tumor cells are strongly correlated with Her2 expression, this result is in agreement with those of Sun and colleagues [21]. However, high B7-H6 + TILs are strongly associated with SBR grade, ER expression and lymph node invasion. Secondly, patients with high B7-H6 tumor expression had worse survival than those with low B7-H6 expression, this result is similar to data from other researchers in both ovarian cancer and breast cancer [18,21]. Nonetheless, patients with high TILs-B7-H6 expression displayed better survival.
Regarding PD-L1 biomarker expression, our data showed that its expression by tumor cells or immune stromal cells has the same clinical signi cance. However, we have noted the strong association of PD-L1 BCC and TILs-PD-L1 with SBR grade. In addition, patient survival curves according to these two biomarkers status display the same trends showing no differences between strong and weak expressions ( Supplementary Fig. 3a-d). These data are consistent with the ndings of some studies, while others have shown the opposite. This point has been reviewed by numerous experts who have justi ed the discrepancy in clinical signi cance of PD-L1 immune checkpoint among published reports [27]. Controversy is argued by the use of different technical supports by different teams and the lack of a complete standardized platform. On the other hand, the great heterogeneity between studied populations may explain also the observed differences.
Further, we seek to explore the relationship between B7-H6 and PD-L1 and their impact on NK cells recruitment. Ours analyzes have shown that there is a strong association between the pro les of PD-L1 in tumor or immune cells with B7-H6-TILs status. In addition, both TILs-B7-H6 and TILs-PD-L1 are highly associated with NK-TILs status. These results consolidate the concept of the mutual crosstalk between tumor cells and immune cells involving different checkpoint molecules which control not only T cell activity, but also NK cells function in a synergistic manner. Knowing that NK cells are associated with better disease outcomes even though they are a minority population in TILs ( Supplementary Fig. 4), these cells play an important role in front of the tumor burden. Additionally, survival analyzes were conducted on different combining groups based on the expression of B7-H6, the main biomarker of this study, with the other immunological parameters that we have chosen to associate which are PD-L1 and NK-TILs statues. Through these analyzes, our data showed that the strong expression of B7-H6 by cancer cells probably leads to a bad progression of the disease among all subgroups. Similarly, but inversely, the high expression of B7-H6 by the immune cells among different subgroups has probably a relation with the awakening process of the immune system involving NK cells and giving the better disease outcomes. This is justi ed by the data of survival curves according to NK-TILs presence in the tumor or surrounds, we have noted that the best group is for patient having the combination of high NK-TILs and TILs-B7-H6 together. Thus, the immune checkpoint B7-H6 and PD-L1 are linked together and certainly with other molecules in a way which modulates the in ammatory tumor microenvironment, we speculate a double role which can polarize the immune response either towards an effective antitumor response or the opposite by maintaining a chronic in ammatory state leading to immunosuppression. Already, different mechanisms damping the function of NK cells have been shown through B7 molecules action. However, there is a lack of knowledge about the expression of these molecules which act differently, indeed their localization whether expressed on the cellular membrane or sequestered in the cytoplasm or even secreted. These concepts justify the complexity in understanding the mechanisms controlling the tumor in ammatory microenvironment through B7 molecules. Taking into account our nding from the present study, we can suspect a protective role of B7-H6 and PD-L1 molecules when co-expressions are manifested on lymphocytes in ltrating tumors, probably these cells produce interleukins such INF-γ which modulate NK cells function. To better elucidate the immunomodulatory mechanism of B7 molecules, it is important to combine more precise analyzes of their location and to conduct analyzes allowing biomarkers co-revelation. Therefore, immunotherapy targeting checkpoint molecules must be revised. The use of combined immune checkpoint inhibitors may result in reduction of tumor burden and patient bene t in some cases. In others situations probably it cannot be effective because of the double role of these two molecules, targeting other molecules from the B7 family can be an important axis which deserves further investigations to decipher their contribution in innate immunity monitoring, probably, we will need to target several molecules at the same time and use targeted therapy according to the patient characteristics.

Patients and tissues samples
Formalin-xed, para n-embedded tissue samples were collected from 156 patients with primary invasive breast carcinomas who underwent surgical resection at the Department of Gynecology and Obstetrics of the Hedi Chaker University Hospital (Sfax, Tunisia). All procedures performed in this study were in accordance with the ethical standards of the institutional and the national research committee of Habib Bourguiba and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All specimens were analyzed at the Department of Pathology of the Habib Bourguiba University Hospital (Sfax, Tunisia) and all tumor tissues were con rmed as the serous breast cancer by using hematoxylin and eosin (H&E) staining after surgical resection. Clinical-pathological data of patients were retrieved from the hospital's electronic records and available paper records. They included age, histological grade, histological type, molecular subtype, tumor size, lymph node status, distant metastasis, and lymphovascular invasion. All tumors were graded according to Elston Ellis modi cation of the Scarff-Bloom-Richardson (SBR) histological grading system [30]. The clinical stage was determined according to TNM (tumor, lymph node and metastasis) classi cation adopted by the International Union Against Cancer [31]. Outcomes data were collected from patient follow-ups at the department of medical oncology of the Habib Bourguiba University Hospital (Sfax, Tunisia). Overall survival (OS) was de ned as time in days from the beginning of the study observation period until death. If the event, death, did not occur, then the OS was noted as the total study observation period in days. Disease free survival (DFS) was de ned from the date of the end of treatment until recurrences or last follow-up.

Immunohistochemistry
Formalin-xed, para n-embedded tissue samples were cut into 5-µm-thick section. Before immunostaining, haematoxylin and eosin-stained standard slides were reviewed by experimented pathologist (S.C) from each section of breast cancer tissues. A representative tumor region and the corresponding formalinxed para n-embedded tissue block were selected for use in the tissue array block containing six fragments of 7 mm in diameter.
In order to validate the immunohistochemical staining for special biomarker, striated muscle and placenta tissues were used as positive controls for the revelation of B7-H6 and PD-L1 respectively according to the instructions proposed by the manufacturers. For immunohistochemistry (IHC), each tumor array was cut into 2-µm sections and mounted on Leica Microsystems BOND Plus slides and dried overnight at 60 °C. Immunohistochemistry steps were performed as described in our previous study [32]. Brie y, tissue slides were depara nized in xylene followed by subsequent rehydration through graded alcohols then washed in puri ed water. Heat-induced antigen retrieval was performed at 95 °C for 20 min in in a pH = 9 epitope retrieval solution (Leica Novocastra) for 40 min. After heating, slides were allowed to cool down to room temperature and were brie y washed in phosphate-buffered saline (PBS) solution.
Immunohistochemical staining was performed using the Novolink Polymer Detection System (RE7150-K, Leica Biosystems). Brie y, endogenous peroxidase activity was neutralized by Peroxidase Block for 5 min. Slides were washed with PBS, followed by application of Protein Block for 30 min. Tumor immunostaining scoring and molecular subtyping The B7-H6 immunostaining densities in tumor cells were analyzed according to the H-score method which has been described in a previous publication [33]. Brie y, H-score was assessed on the basis of the percentage of positive tumor cells with clearly brown cytoplasm and/or membrane immunostaining. H-score was calculated as follows = (% tumor cells unstained x0) + (% tumor cells stained weak x1) + (% tumor cells stained moderate x2) + (% tumor cells stained strong x3), and it ranged from 0 (100% negative tumor cells) to 300 (100% strong staining tumor cells). B7-H6 expression was dichotomized into two groups according to the frequency distributions of the H-scores displaying the median as the cut-off value (H-score 0-99 = negative/low expression, and 100-300 = positive/high expression). Frequency and staining intensity of PD-L1 by tumor cells were analyzed, and PD-L1 expression was quanti ed using the H-score method according to the previous publications [26 , 27].
Breast cancer molecular classi cation is based on the expression of classical biomarkers including estrogen (ER) and progesterone (PR) receptor, the human epidermal growth factor receptor 2 (Her2) and Ki-67 labeling index as a cell proliferation biomarker. Expression of all biomarkers was carried out using immunohistochemical method.
Statistical analyses were performed using the R language and the SPSS 20.0 statistical software for Windows (SPSS Inc., IBM). Bivariate analysis was performed to assess the correlation between biomarkers and clinicopathological characteristics using Chi-square tests for qualitative variables, Pearson-rank correlation for quantitative variables and ANOVA test for quantitative variation among classes of qualitative variables. The cumulative survival (overall survival, OS; recurrence-free survival, RFS) times were calculated using the Kaplan-Meier method and compared with the log-rank test. A cox-regression was also performed in order to evaluate the signi cance of prognostic factors on survival in a multivariate context (adjusting for confounding variables). All pvalues less than 0.05 were considered signi cant.    showing NK-TILs distribution according to TILs-PD-L1 status (g, p < 0.01) and TILs-B7-H6 status (h, p < 0.001). Barplot representation of TILs-B7-H6 distribution among TILs-PD-L1 status (I, p < 0.001).